Purpose: Multidrug-resistant TB (MDR-TB) has been reported in almost all parts of the world. Childhood TB is accorded low priority by national TB control programs. Probable reasons include diagnostic difficulties, limited resources, misplaced faith in BCG and lack of data on treatment. Good data on the burden of all forms of TB among children in India are not available. Objective: To study the drug sensitivity pattern of tuberculosis in children aged from 3 months to 18 years and the outcome of drug-resistant tuberculosis by BACTEC culture system and PCR-based DNA sequencing technique. Materials and Methods: This is a retrospective study. One hundred and fifty-nine clinical specimens were processed for Ziehl-Neelsen stain, Mycobacterial culture by BACTEC method, phenotypic DST for first-line drugs for Mycobacterium tuberculosis (M. tuberculosis) isolates and PCR-based DNA sequencing was performed for the M. tuberculosis isolates targeting rpoB, katG, inhA, oxyR-ahpC, rpsL, rrs and pncA. Results and Conclusion: Out of the 159 Mycobacterial cultures performed during the study period, 17 clinical specimens (10.7%) were culture positive for M. tuberculosis. Among the 17 M. tuberculosis isolates, 2 were multidrug-resistant TB. PCR-based DNA sequencing revealed the presence of many novel mutations targeting katG, inhA, oxyR-ahpC and pncA and the most commonly reported mutation Ser531Leu in the rpoB gene. This study underlines the urgent need to take efforts to develop methods for rapid detection and drug susceptibility of tubercle bacilli in the pediatric population.

Annual rate of tuberculosis infection is a most sensitive indicator of prevalence of TB infection in a community. India has the highest TB burden country accounting for 1.9 million cases of the 9.1 million cases of global incidence. [1] Multidrug-resistant TB (MDR-TB) [2] has been reported in almost all parts of the world. Several small surveys conducted across India have shown prevalence rates of MDR-TB in the country at around 3% among new cases and 12% among retreatment cases. [3],[4] Global estimates have reported 1.5 million new cases of tuberculosis in children [5] and nearly 8-20% of the deaths caused by tuberculosis occur in children. [1] Childhood TB is accorded low priority by national TB control programs. Probable reasons include diagnostic difficulties, limited resources, misplaced faith in BCG and lack of data on treatment. [2] Good data on the burden of all forms of TB among children in India are not available. Hence an attempt was made to find out the drug sensitivity pattern of Mycobacterium tuberculosis (M. tuberculosis) isolates from a tertiary referral center for children in Chennai.

~ Materials and Methods

Clinical specimens

This project was approved by the Institutional (Vision Research Foundation, Chennai, India) Ethical Committee. The clinical specimens received from 159 patients referred by pediatricians from KKCTH for isolation of M. tuberculosis by BACTEC culture method during the period April 2007-July 2009 were included in the study for analysis of results. The pediatricians provided the following clinical details from medical records of the patients - age, sex, clinical diagnosis with type of tuberculosis, contact history, nutritional status, mantoux test, chest X-ray, treatment regimen and outcome of the treatment.

Mycobacterial culture

Mycobacterial culture was performed by BACTEC TB 460 reader after standard decontamination with NALC-NaOH following the manufacturer's instruction [6] (BD, India). After isolation, the isolate was confirmed as M. tuberculosis by NAP test [6] and also by PCR targeting MPB64 [7] gene and IS6110 [8] region. Phenotypic drug susceptibility testing for first line anti-tuberculosis drugs (Streptomycin, Isoniazid, Rifampicin, Ethambutol and Pyrazinamide) was performed by BACTEC TB 460 reader. [6] Antibiotic stock solutions for streptomycin, isoniazid, rifampicin, ethambutol and pyrazinamide were prepared and kept in -20˚C until use and the final concentrations used were 4.0 μg/ml, 0.2 μg/ml, 2.0 μg/ml, 5.0 μg/ml and 100 μg/ml, respectively.

DNA was extracted from all the 17 M. tuberculosis isolates by keeping the suspension at 80°C for 10 minutes. PCR was carried out targeting the following genes to detect the mutations conferring resistance: rpoB for rifampicin, katG, inhA and oxyR-ahpC intergenic region for isoniazid, rpsL and rrs for Streptomycin and pncA for pyrazinamide. [Table 1] lists the sequences of the different primers used in the study with their positions, thermal profile and the amplified product size. The amplified product was subjected to electrophoresis on 2% agarose gel incorporated with 0.5-μg/ml ethidium bromide for visualization by UV transilluminator (Vilber Lourmet, France).

Table 1: Primer sets (Source: Bangalore Genei, India) used in the study to sequence the different loci of target genes with their thermal profile, expected amplicon sizes and nucleotide sequence of amplicons

DNA sequencing of amplicons was carried out using an ABI prism 3110 automated DNA sequencer (Applied Biosystems, USA). Cycle sequencing of the amplified products was carried using BigDye terminator kit (ABI Prism, USA) following manufacturer's instruction. The sequences were analyzed by sequence analysis software Bio Edit sequence alignment software. The sequences generated were compared with the wild type sequence by using Multalin software to identify the presence of mutation or polymorphism.

Standard precautions for PCR

Adequate and rigorous precautions were taken to prevent amplicon contamination. Separate rooms were used for the preparation of DNA, its amplification and analysis of amplified products. PCR cocktail were prepared in a laminar flow workbench and the microfuges tubes and milliQ water used for PCR were double sterilized.

~ Results

Mycobacterial culture and phenotypic drug susceptibility testing

The age group of the 159 clinical specimens included in the study ranged from 3 months to 18 years. Of the 159 mycobacterial cultures performed, 17 (10.7%) were culture positive for M. tuberculosis. Phenotypic drug susceptibility testing was performed for all the 17 M. tuberculosis isolates (11 respiratory specimens, 7 CSF, 1 pus aspirate and 3 gastric aspirates). Direct smear was positive for AFB in 7 specimens (BAL-3, Sputum-2, and Gastric aspirate 2). Out of the 17 M. tuberculosis isolates, two isolates were from the same child from two specimens, gastric juice (3695/09) and bronchoalveolar lavage (BAL) (3753/09). Chest X-ray was abnormal in eight children, showing collapse, consolidation in all and peribronchial infiltrates in two. Contact history was established in 5 (38.4%) children, 5 (38.4%) children were mantoux test positive and 4 (30.7%) children were severely malnourished [Table 2].

Among the 17 M. tuberculosis isolates, 7 were sensitive to all the 5 first-line drugs and 10 were resistant to either one or more than one drug [Table 2]. Among the 10 resistant isolates, 7 were monoresistant [2 to isoniazid (2033/07, 2636/08) and 5 to pyrazinamide (0902/09, 2966/09, 3227/09, 3695/09 and 3753/09)], 1 was resistant (3744/09) to 3 drugs (isoniazid, ethambutol and pyrazinamide) and 2 were MDR-TB (4610/07, 0327/09). One MDR-TB isolate (from BAL specimen of 18-year-old female-4610/07) was resistant to all the 5 first-line drugs and the other one (from a 5-month-old male - 0327/09) was resistant to isoniazid, rifampicin, pyrazinamide and streptomycin. Both MDR-TB specimens were negative for AFB in the direct smear. None of the isolates were monoresistant to rifampicin and streptomycin except the two MDR-TB isolates (4610/07, 0327/09).

All the above 10 children were started on antituberculosis chemotherapy as per IAP (Indian academy of Pediatrics) and RNTCP (Revised National Tuberculosis Control Program). Among the above two MDR-TB patients, the first patient died but there was no contact history given, whereas the second patient's maternal grandfather had a history of tuberculosis and the child is doing well with regular follow-up. Interestingly majority of the isolates (5/10) were monoresistant to pyrazinamide, followed by isoniazid (2/10).

~ PCR-based DNA sequencing results

rpoB

All the 17 M. tuberculosis showed a specific band at 350 bp. The sensitivity of the PCR was 10 fg and it was specific to detect M. tuberculosis H37Rv and H37Ra DNA. The nucleotide sequence of resistant isolate was compared with rifampicin-sensitive M. tuberculosis isolates, positive control M. tuberculosis H37Rv ATCC and reference strain from genbank accession No. L27989. Phenotypically rifampicin sensitive M. tuberculosis isolates did not show any mutation. Ser531Leu, the most common reported mutation which codes for rifampicin resistance was detected in both the MDR isolates (4610/07, 0327/09).

katG

PCR targeting two different regions of katG (3-239 and 1187-1600) gene of M. tuberculosis H37Rv was standardized using specific forward and reverse primers. The sensitivity of the PCR was 10 fg and 85 fg for primer set 1 and 2, respectively, and it was specific to detect M. tuberculosis H37Rv and H37Ra DNA.

Among the 17 M. tuberculosis isolates, one MDR-TB isolate did not amplify for the both the katG regions (4610/07). The remaining 16 isolates showed a specific band at 237 bp and 414 bp region for primer set 1 and primer set 2, respectively. All the mutations targeting katG gene are novel in this study. In the katG 1 region, deletion of nucleotide 'C' at position 13 was detected in all the 4 isoniazid-resistant strains [2 INH monoresistant (2033/07, 2636/08), 1 polyresistant and 1 MDR-TB)]. In addition to the deletion mutation, insertion of nucleotide 'T' at position 24 was detected in isoniazid-monoresistant strain from BAL (2636/08) and 2 substitution mutations [AAC→ACC (Asn35Thr), CTG→CGG (Leu48Arg)] were detected in MDR-TB isolate (0327/09) from BAL [Table 3].

All the 17 sensitive and resistant strains of M. tuberculosis isolates showed a specific band at 248 bp except one resistant XVisolate from BAL. Phenotypically isoniazid sensitive isolates did not show any mutations. Isoniazid-monoresistant strain from CSF (2033/07) showed the presence of 4 novel substitution and 3 novel silent mutations [Table 3]. One novel deletion mutation of nucleotide T at position 83 was detected in 2 isoniazid-resistant strains [1 polyresistant (3744/09) and 1 MDR (0327/09)]. In an isoniazid-monoresistant strain from BAL (2636/08), one novel silent mutation was found at codon position 63 (CT C to CT T - Leu63Leu).

oxyR-ahpC

All the 17 sensitive and resistant strains of M. tuberculosis isolates showed a specific band at 701 bp except one MDR isolate from BAL. The nucleotide sequence of resistant isolate was compared with sensitive strain of M. tuberculosis, positive control M. tuberculosis H37Rv ATCC and reference strain from genbank accession No. MTU16243. There were no mutations in the 2 isoniazid-monoresistant (2033/07, 2636/08) and 1 polyresistant (3744/09) M. tuberculosis isolates. One MDR isolate from BAL (0327/09) showed the presence of 8 novel substitution and 2 novel silent mutations [Table 3].

rpsL and rrs

The nucleotide sequence of resistant isolate was compared with sensitive strain of M. tuberculosis, positive control M. tuberculosis H37Rv ATCC and reference strain from genbank accession No. X70995. Among the 2 Streptomycin-resistant isolates, one MDR-TB isolate did not amplify (4610/07) and the other - MDR-TB isolate (0327/09) had one substitution mutation in the 129 position (AAG to AGG - Lys43Arg) of rpsL gene which is reported earlier. The streptomycin sensitive strains and positive control did not show any mutation. In the rrs gene, there were no mutations.

pncA

PCR targeting the pncA gene was standardized using specific forward and reverse primers. The sensitivity of the PCR was 163 pg of M. tuberculosis DNA and it was specific to detect M. tuberculosis H37Rv, M. tuberculosis H37Ra and M. bovis DNA. Out of the 17 M. tuberculosis isolates, one MDR isolate (4610/07) did not amplify and the remaining 16 isolates showed a specific band at 670 bp. Among the 5 pyrazinamide-monoresistant isolates, 4 isolates (2966/09, 3227/09, 3695/09, 3753/09) did not show the presence of any mutation and one monoresistant isolate (0902/09) showed the presence of novel substitution mutation at the 179 th codon position ( A GCto T GC-Ser179Cys). In one MDR isolate from BAL (0327/09), there were 11 novel substitution mutations [Table 4].

The MDR-TB (0327/09) isolate from BAL specimen which is phenotypically resistant to rifampicin, isonazid, streptomycin and pyrazinamide showed the presence of mutation in all the genes except rrs gene.

Tuberculosis remains an important and potentially preventable cause of childhood illness and death. Children have a high risk of progression to disease following infection, and are much more likely to develop severe or disseminated tuberculosis. Children with latent tuberculosis infection (LTBI) become the reservoir of future disease in adulthood, perpetuating the epidemic. Accurate data on childhood tuberculosis is not available due to the inherent difficulties in the diagnosis in children. Until recently most National Tuberculosis programs (NTPs) did not collect data on childhood tuberculosis by age and clinical syndrome, reflecting its perceived lower priority. [12]

India is ranked 17 th among the 22 high burden countries in terms of TB incidence rate. [13] The total prevalence of MDR-TB is about 3% in new cases, and 12-17% in retreatment cases. [13] In a recent study by Swaminathan et al., [14] the prevalence of isoniazid resistance was estimated as 12.6% and MDR-TB as 4% in children. The RNTCP status report 2009 states that nearly 40% of the Indian population is infected with the TB bacillus and 10% of new cases of occur in pediatric population. [13] Annual rate of tuberculosis infection is most sensitive indicator of prevalence of TB infection in a community. For adequate control of tuberculosis, the average ARTI needs to be 0.07% below 14 years of age. A nationwide survey among young children show a very high figures of ARTI in almost all the regions-highest in North zone (1.9%) followed by West Zone (1.8%), East Zone (1.3%) and South zone (1.1%). The results indicate a high rate of transmission of infection due to high load of infectious cases in the community. [15]

In the present study, among the 17 M. tuberculosis isolated from different clinical specimens, only 7 were positive for acid-fast bacilli in the direct smear, and the two MDR isolates in this study were smear negative, emphasizing the need for culture, even if direct smear was negative. It is alarmingly dangerous fact that 20% (2/10) of the M. tuberculosis strains are MDR in this study. Out of the 10 children with drug-resistant tuberculosis, 3 children died, one due to multiple tuberculoma, second one with respiratory failure and third with pulmonary alveolar proteinosis. The remaining 5 children are doing well with regular follow-up.

In the isoniazid-resistant isolates, one novel deletion mutation of nucleotide "C" at position 13 was detected in all the 4 isonaizid-resistant isolates targeting katG gene. There were 8 novel substitution mutations, 1 novel silent and one novel insertion mutation targeting katG I and II region. These mutations were detected in isolates from various anatomic sites like CSF, BAL and pus aspirate from spine. As reported in the previous studies, the most commonly found mutation Arg463Leu in the katG gene of isoniazid sensitive and resistant isolates was not found in the present study. [9],[10] In the inhA gene, there were 4 novel substitution and 3 novel silent mutations. In oxyR-ahpC intervening region, there were 8 novel substitution and 2 silent mutations. When comparing the relative distribution of mutation in the 3 target loci katG and inhA gene revealed that 3 isolates had mutation in both katG and inhA gene and only one MDR isolate from BAL had mutation in all three target loci.

The Lys43Arg reported substitution mutation targeting rpsL gene which results in high level of streptomycin resistance was detected in only one MDR isolate from BAL. No mutations were detected in the rrs gene of Sterptomycin. However, we did not find the silent mutation in the 43 codon (Lys43Lys) as reported in earlier studies. [10],[11]

Novel mutations targeting pncA gene was detected in only 2 PZA-resistant (1 monoreistant and 1 MDR-TB) isolates. In this study, 6 M. tuberculosis isolates that were phenotypically resistant did not show the presence of any mutation. There may be an alternative mechanism for the PZA resistance related to the efflux pump of pyrazinamide rather than the mutation in pncA gene causing the drug resistance. There were reports in literature that 70% of the phenotypically resistant isolates did not show mutations after genotypical analysis. [16]

Nearly two-third of M. tuberculosis in this study are found resistant to the first-line drugs highlighting the need to apply rapid diagnostic tests for the detection of drug-resistant tuberculosis for effective treatment and thereby prevent the spread of tuberculosis in the community. The limitations of the study are that contact history is not available for all cases included and the study results may not exactly represent the prevailing situation in the community as referred patients only are seen in the tertiary care centre.

The study is an eye opener on the high level of drug-resistant M. tuberculosis strains circulating in pediatric population. Further it emphasizes the utmost importance of culture and sensitivity for appropriate treatment to prevent the spread of drug-resistant strains in the community.